Introduction

This Page contains all the information regarding DNA-Overstretching experiments and results.

Proof of DNA-tethering

Two video presents good DNA-tethers in H2O and D2O. For some reason D2O tethers look little better. Each moving bead has a ds-DNA tether which is roughly 1500nm long (4.4kb; 110ng/ml; 02/11/11). The bead size is 530nm diameter. This sample is prepared in water and heavy water for DNA-overstretching experiments which i will discuss below.

Experiments

Experiment 1 (01/04/2013)

Prepare some new buffers and attempt DNA-tethering in H2O and D2O with 265nm beads (bead radius).

Buffer preparation

BGB is good only for few weeks and it has been over 2 months since i made it last, so i am going to make new BGB and antidig. For more information go to Buffer preparation for DNA overstretching and unzippingexperiments[1]

Data

Experiment 3 (01/07/2013)

FTC was fixed for big beads. Find tether center (FTC) is a step sequence program which finds a tether center, centers the trap and acquires data for repeatable geometer. This experiment I used big beads (radius 520 nm).

Result:

Sample was very successful lot of good tethers and sample was still good more than over 3 hour period.

The overstretching data looks really good the overstretching force is at 65pN.

FTC was fixed and working great.

Data

Data acquisition parameters: DNA4.4kb(110ng/ml; 02/11/11) ,r=265nm ,medium h2o,TCH/BH 574nm,FH 200 & 300nm. The best focal height is 300nm which gives TCH/BH of 674 nm. But this data was taken at TCH/BH of 574nm. This height is not too height or too low.

Overstretching-data and other information can be seen through evernotes:

Experiment 6 (01/15/2013)

I did two overstretching experiments today with H2O and D2O with 4.4kb (110mg/ml;02/11/11) DNA. From tomorrow i will be using new DNA so i think this is the last experiment i have with this DNA, since it is getting old now.

Overstretching in H2O and D2O worked fine but D2O was lot better than H2O.

Procedure

The procedure is same as experiment 5(01/10/13)

Result:

Sample is very successful tethering wise, lot of tethers, but most of them broke during the beginning of overstretching after a good FTC. Very very few tethers overstretched; 11 good tethers out of 75 attempts. I selected some of the beast segments; force goes from 59 to 66pN at 1750nm LDNA at which the DNA starts overstretching. The highest force is the green curve which i think should not belong to this data because the FTC has a huge nick at the center which was very unusual but rest of the data is good with the error range of stiffness. This data is analyzed under quick conversion, because we have not done the geometry going so the data might change.

Some tethers overstretched multiple times, i noticed every time the same tethers is overstretched the overstretching force depletes. it is not quite noticing in some tethers but on average it sounds true.

Repeated overstretching segments (the segments in which same tether overstretched multiple times) are:(0000-(3,4),(10,11,12)); first number shows the overstretch first time consecutive ascending number shows 2nd, 3rd, so-forth and so-on time overstretch.